5 September 1970 MIDICALBarrJOURNL 559 Preliminary Communications

Papillary Necrosis in Rats Caused by hours' overnight dehydration daily. After eight weeks of anal- gesic feeding one rat from each analgesic group was killed Aspirin and Aspirin-containing Mixtures each week over the next 12 weeks. A proportion of animals from group 1, Table I, and the control group were subjected British Medical journal, 1970, 3, 559-561 to oligaemic shock by removal of 2 to 3 ml. of blood by car- diac puncture under light anaesthesia. These animals were Summary: Nearly half the rats gavage-fed with aspirin kept on dehydration for 48 hours before killing. At the end of and aspirin-containig mixtures developed papillary the trial the animals were killed and the kidneys removed for necrosis in 20 weeks. This incidence is similar to that histological examination. found in rats on A.P.C. mixtures with high and low con- centrations of p-chloracetanilide, an impurity of TABLE I.-Analgesic Mixtures Used in Trial 1 phenacetin. Aspirin alone produced necrosis in 7 out of 19 rats whereas phenacetin in the same dose had (36-8%) AnalgesicAnalgesic a (mg./kg./day)Dose NoRatsof failed to cause any renal damage over six to nine months. If these results also apply to man they suggest that Group 1: A.P.C. (high impurity) .900 (A = 380) 40 Group 2: A.P.C. (low impurity) 900 (A = 380) 10 aspirin and not phenacetin may be the major factor in Group 3: A+P .750(A=380) 10 analgesic nephropathy in patients taking A.P.C. mixtures. Group 4: A+C .700(A=500) 10 Group 5: A 500 20 An augmented clearance of aspirin appeared to afford Group 6: Control . . 20 some protection to the medulla, and it is suggested that this observation may have important clinical and A = Aspirin. P = Phenacetin. C = Caffeine. epidemiological applications. Trial 2.-In this trial 80 female nulliparous Sprague- Dawley rats were used. The analgesic mixtures were adminis- INTRODUCTION tered for 20 weeks as in the first trial, the analgesics used being shown in Table II. Fifty rats were sustained on a con- Spiihler and Zollinger (1953) first drew attention to the asso- stant water diuresis with 5 % glucose as drinking-water ciation between phenacetin and interstitial nephritis. There is (Andriole and Epstein, 1965). Thirty rats were subjected to now considerable evidence that prolonged intake of analgesics dehydration as in the first trial and simultaneously given is associated with a form of renal disease characterized by acetazolamide and sodium bicarbonate with the analgesics. papillary necrosis (Hultengren, 1958; Lindeneg et al., 1959; Half the rats from this group were also subjected to oligae- Burry et al., 1966; Dawborn et al., 1966; Gault et al., 1968). mic shock as in the previous trial. The animals were killed Phenacetin, though never prescribed alone, has been singled weekly over a period of 8 to 20 weeks. out and blamed for this lesion because it is the common factor in analgesic mixtures (Prescott, 1966; Kincaid-Smith, TABLE II.-Analgesic Mixtures Used in Rats in Trial 2 1967; Shelley, 1967). The other analgesics often prescribed with phenacetin are aspirin, caffeine, amidopyrine or its Analgesic Analgesic 1 (mg./kg./day)~~~Dose No.Ratsof analogues, and phenazone. All of them have been shown to be nepbrotoxic in animals and humans (Wendt, 1938; Axel- Rats on a Constant Water Diuresis with 5% Glucose Group 1: A.P.C. 900 (A = 380) 10 sson, 1958; Boyd, 1959; Fazekas et al., 1960; Clausen, 1962, Group 2: A+NAPA+C .900 (A = 380) 10 et 1964; Fellers et al., Group 3: A+C .700 (A=500) 10 1964; Harvald, 1963; Abrahams al., Group 4: A 500 10 1965; Prescott, 1965, 1969; Brown and Hardy, 1968). Group 5: Control . 10 Saker and Kincaid-Smith (1969) showed that rats gavage- Rats on 16 Hours' Dehydration a Day fed with an aspirin, phenacetin, and caffeine (A.P.C.) mixture Group 1: A.P.C. +acetazolamide (20 mg./kg./day) .. 900 (A = 380) 10 Group 2: A+ acetazolamide (20 mg../kg./day) .. .. 400 10 in a dose taken by some patients with analgesic nephropathy Group 3: A+sod. bicarb. (50 mg./lkg./day) .. .. 500 10 will develop renal papillary necrosis in six to nine months. Phenacetin in twice the dose contained in the A.P.C. mixture A = Aspirin. P = Phenacetin. C = Caffeine. NAPA = Paracetamol. failed to cause any renal damage, suggesting that some factor other than phenacetin in A.P.C. was responsible for papillary Serial renal function tests including an assessment of necrosis. To study the cause and pathogenesis of analgesic medullary blood flow were performed in some rats. The nephropathy we have extended the work of Saker and Kin- results of these studies and details of the pathological find- caid-Smith in an attempt to define the ingredient of A.P.C. ings will be published separately. mixtures which causes papillary necrosis.

PATHOLOGICAL DEFINITIONS MATERIAL AND METHODS Histological grading was done on a blind basis. Trial 1.-Sixty Sprague-Dawley and fifty Wistar female The kidneys were included as papillary necrosis in Tables nulliparous rats, equally distributed between groups of rats, III and IV when they showed frank necrosis, either diffuse or were fed analgesics for 20 weeks in two separate studies. One patchy, in the papillae. Diffuse papillary necrosis (see Fig.) of these trials was conducted during the winter and the other showed total necrosis of the papillae with a clear line of during summer. The analgesic mixtures were given by gavage demarcation between necrotic and viable renal tissue. The twice daily, five days a week. The number of animals in the patchy medullary lesion showed either necrosis of loops of two studies and the analgesic mixtures used are shown in Henle and vasae rectae with some surviving collectng ducts Table I. The maximum dose of the drug administered was or areas of necrosis of all elements with intervening areas of achieved over two weeks by progressively increasing the dose surviving tissue. Minor degrees of change including every two to three days. All animals were subjected to 16 necrobiosis are excluded for the purposes of this report. 560 5 September 1970 Preliminary Communications, TABLs III. ncidence of Renal Papillary Necrosis with Aspiri ining Mixtures over 8 to 20 Weeks-Rats on 16 Hours' D.dracion a Day out of 19 rats (36-8%). This incidence is not significantly different from that in the A.P.C. groups. In spite of a higher Analgesic No. of Rats Papillary Necrosis dose of aspirin, a combination of aspirin and caffeine produced necrosis in only 10% of rats compared with 40% in Control 19 0 rats fed on with A.P.C. (high impurity) 31 13 (41-9%) aspirin combined phenacetin. A.P.C. (low impurity) .10 6 (60%) A constant water diuresis or the administration of aceta- A+P 10 4 (40%) A+C 10 1 (10%) zolamide or sodium bicarbonate- appeared to reduce the A (500 mq./kg./day) 19 7 (36 8%) of mixtures on the renal *Phenacetin (500 mg./kg./day) .27 0 (over 6-9 months) effect analgesic medulla (Table IV). The incidence of papillary necrosis was 38-8% in the de- with in rats on a constant *From Saker and Kincaid-Smith (1969). hydration group compared 16-7% water diuresis and 10-7% in rats having added acetazolamide and sodium bicarbonate. TABs IV.-Incidence of Renal Papillary Necrosis with Aspiin-contaming Oligaemic shock was induced in 58.6% of analgesic-fed rats Mixtures over 8 to 20 Weeks and in 69% of control rats befor, killing; 21.9% of the anal- The incidence Analgesic No. of Papillary Necrosis gesic-fed animals died spontaneously. of papillary necrosis in analgesic-fed rats on dehydration Rats on a Constant Water Diuresis with 5% Glucose subjected to oligaemic shock (51.6%) was higher than in Control . . 10 0 A.P.C. .9 0 animals dying spontaneously (45.5%) or those killed without A+NAPA+C .. 9 2 (22 2%) but these were not A+C 9 1 (11-1%) cardiac puncture (37.5%), differences A..9 3 (33-3%) statistically significant. Rats on 16 Hours' Dehydration a Day A.P.C. +acetazolamide ..8 1 (12-5%) A. +aczolamide .10 0 A+sodium bicarbonate .10 2 (20%) DIscussIoN In the past, renal papillary necrosis has been produced in animals with both aspirin and phenacetin. The very high doses used in these experiments have been strongly criticized (Gilman, 1964). Abrahams et al. (1964) and Saker and Kin- caid-Smith (1969) gave doses of A.P.C. to rats similar to those taken by some patients with papillary necrosis and produced a renal lesion identical to that seen in patients with analgesic nephropathy in nearly 50% of animals. In an attempt to shorten the period of feeding before necrosis develops we have in our present study doubled the dose of analgesics and induced necrosis in 8 to 20 weeks. The short- term feeding has enabled us to study a greater number of combinations of analgesics and to identify the probable toxic agent in analgesic nephropathy. Renal papillary necrosis was observed in the same per- centage of rats fed aspirin alone as that found in rats given an A.P.C. mixture over 8 to 20 weeks. Phenacetin alone in the same dose over nine months failed to cause any renal damage (Saker and Kincaid-Smith, 1969). On the basis of these studies it seems likely that aspirin is the main factor causing papillary necrosis in rats fed on A.P.C. mixtures. For many years salicylates have been known to cause renal Necrotic rat papilla showing. outlines of necrotic tubules. The darker- staining areas are calcified material. The changes are similar to those seen damage, and there have been both clinical and experimental in analgesic nephropathy in man. ( x 210.) reports of a variety of lesions, including haematuria and renal failure (Hanzlik, 1927; Lipman et al., 1949; Locket, 1957; Campbell and Maclaurin, 1958; Granville-Grossman and Ser- RESULTS geant, 1960; Parsons, 1963; Scott et al., 1963; Ben-Ishay, 1964; Prescott, 1965; Dubach and Josch, 1967; Robinson et The kidneys from 19 rats were excluded from evaluation al., 1967). Renal papillary necrosis has been produced in because of early deaths from and aspiration pneumonia animal experiments with aspirin alone (Clausen, 1962, 1964; gastrointestinal ulceration and bleeding or advanced autolytic Fellers et al., 1965; Robinson et al., 1967). Though aspirin is changes. commonly taken with other analgesic compounds, cases of The combined results of the two studies conducted in Trial analgesic nephropathy and papillary necrosis with aspirin 1 are shown in Table III. Over a period of 8 to 20 weeks on alone have also been reported (Harvald, 1963; Parsons, 1963; aspirin-containing mixtures nearly half the rats developed Gault et al., 1968; Prescott, 1969). necrosis. The animals fed on A.P.C. in the summer study in Patients with rheumatoid arthritis are an important group this trial showed a higher incidence of frank and -severe in whom the clinical association between aspirin ingestion papillary necrosis (69.2%) compared with those in the winter and papillary necrosis may be further studied. Most patients study (22-2%). This appeared to be related to the fact that in with rheumatoid arthritis consume large quantities of anal- the summer study the rats were often exposed to prolonged gesics, especially salicylates. The incidence of papillary periods of hot and dry weather and the most severe forms of necrosis and interstitial nephritis in necropsy and renal necrosis were seen in rats which died spontaneously on hot biopsy surveys in patients with rheumatoid arthritis is high. days. There were no facilities to maintain the temperature of The incidence in different reports has varied from 21-3% to the animal-room at a constant level. The incidence of necrosis 28.1% (Clausen and Pedersen, 1961; Brun et al., 1965; Law- in rats fed on a proprietary A.P.C. mixture containing high son and Maclean, 1966; Bulger et al., 1968). In a necropsy sur- impurity content (0-1% p-chloracetanilide) is similar to that in vey conducted by us at the Royal Melbourne Hospital a 100% rats on a relatively pure A.P.C mixue (01% p-chlor- incidence of papillary necrosis was found in eight patients acetanilide). Aspirin alone produced papillary neis in 7 coming to necropsy during the one-year period of survey 5 September 1970 Preliminary Communications B 561 (Nanra et al., 1970). This very high incidence of papillary REFERENCES necrosis in rheumatoid arthritis in our hospital has been fur- Abrahams, C., Rubenstein, A. H., Levin, N. W., and Wunderlich, U. nine coming to necropsy (1964). Archives of Pathology, 78, 222. ther confirmed in another patients Andriole, V. T., and Epstein, F. H. (1965). 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